3.2 Assembling caprolactone-producing strains
To assess CDH and CHMO gene expression to different levels, we generated
two ε-CL producers based on the platform organism for Cyp gene
expression developed recently [18]. First, CDH and
CHMO genes were placed downstream of the Cyp genes on the same operon inP. taiwanensis VLB120 pSEVA_CL_1 (Figure 3A). Consequently, one
mRNA is produced, harboring all 5 genes sequentially. To enhance CDH and
CHMO levels, a second strain harboring pSEVA_CL_2 was created.
pSEVA_CL_2 contains a second Ptrc promoter
upstream of the CDH and CHMO genes giving rise to increased expression
rates of the respective genes.
In bioconversions applying resting P. taiwanensis VLB120
(pSEVA_CL_1), ε-CL accumulated up to 1.46 ± 0.01 mM within 120 min,
after which the reaction was stopped (Figure 3B). Besides the desired
product ε-CL, also the intermediate cyclohexanol was detected to a
maximal concentration of 42 µM after 60 min. Additionally, 6HA, the
hydrolysis product of ε-CL (Figure 1), accumulated in the culture
(especially in the second hour of bioconversion) and reached a final
concentration of 0.77 ± 0.07 mM after 120 min. The specific overall
product formation rate considering ε-CL and 6HA remained quite stable at
a high level (37.3 ± 1.9 U gCDW-1).
The same experiment employing P. taiwanensis VLB120
(pSEVA_CL_2) (Figure 3D), resulted in ε-CL accumulation to a 20 %
higher concentration of 1.80 ± 0.01 mM after 120 min and a higher
specific product formation rate
(43.4 ± 1.9 U gCDW-1). In contrast to
pSEVA_CL_1, the insertion of the second promoter completely prevented
the emergence of cyclohexanol, whereas 6HA accumulated to a comparable
concentration of 0.7 mM within 120 min. The activity increase observed
in the first 10 min of both experiments (Figure 3BD) may be attributed
to the direct addition of liquid cyclohexane into the bacterial culture
resulting in high local and thus toxic/inhibitory cyclohexane
concentrations, which then were attenuated upon cyclohexane
redistribution among gas and liquid phase.
The direct comparison of both strains carrying either pSEVA_CL_1 or
pSEVA_CL_2 via SDS-PAGE showed that Cyp levels were similar (Figure
3CE). CDH and CHMO levels were close to the detection limit in P.
taiwanensis VLB120 (pSEVA_CL_1), whereas the insertion of the second
promoter in the construct pSEVA_CL_2 significantly enhanced CDH and
CHMO levels (Figure 3E). Assessing the initial specific activities of
pSEVA_CL_1 containing enzymes for cyclohexane
(37 U gCDW-1), cyclohexanol
(39 U gCDW-1), and cyclohexanone
(44 U gCDW-1) conversion revealed
similar values for all three reaction steps (Table 2) with the CHMO
activity being slightly higher than the other two. The higher CDH and
CHMO content of P. taiwanensis VLB120 (pSEVA_CL_2) directly
translated into higher alcohol
(76 U gCDW-1) and ketone
(84 U gCDW-1) conversion activities,
respectively (Table 2). The introduction of the second promoter doubled
the CDH and CHMO activities without affecting the amount of active Cyp
in the cells (Table S4). Coexpression of CDH and CHMO together with Cyp
genes resulted in a 20 % growth rate reduction from 0.37 ± 0.01
(pSEVA_Cyp) to 0.29 ± 0.01 h-1 (pSEVA_CL_1),
indicating a metabolic burden (Table S4). Concomitantly, the active Cyp
content decreased by 30 %. Interestingly, such decreases in growth rate
and active Cyp content were not observed with pSEVA_CL_2 (Table S4).
These results indicate that higher CDH and CHMO levels are crucial to
prevent the accumulation of cascade intermediates, especially of the
CHMO inhibitor cyclohexanol, and thus to drive the cascade towards ε-CL
formation. Furthermore, the two-operon approach reduced the metabolic
burden as indicated by the growth rate of the respective strain compared
to the one-operon approach.
To further characterize cyclohexanol conversion efficiencies, different
cyclohexanol concentrations were added to P. taiwanensis VLB120
cells containing pSEVA_CL_1 or pSEVA_CL_2 (Figure 3F). With
pSEVA_CL_1, increasing cyclohexanol led to a decrease in the initial
specific ε-CL formation rate and the accumulation of cyclohexanone in
the culture (Figure 3F). This correlated with CHMO inhibition and only
15 % of the produced cyclohexanone were converted to ε-CL when 1 mM of
cyclohexanol was added as substrate. For a similar cyclohexanol amount
(1mM), the elevated CDH and CHMO levels in cells carrying the
pSEVA_CL_2 construct resulted in a stable activity of the overall
cascade, giving rise to higher cyclohexanol and, subsequently,
cyclohexanone conversion with 35 % being converted to ε-CL (Figure 3F).
Cyclohexanone accumulation was only observed for initial cyclohexanol
concentrations of ≥ 0.4 mM.
In conclusion, both tested strains exhibited decent specific whole-cell
activities for the entire cascade. The main difference consisted in the
production of small amounts of cyclohexanol with pSEVA_CL_1. Due to
CHMO inhibition and CDH kinetics, cyclohexanol was found to potentially
disrupt the cascade in a self-enforcing manner. However, the high CDH
and CHMO expression levels in P. taiwanensis VLB 120
(pSEVA_CL_2) efficiently prevented cyclohexanol accumulation.
Furthermore, the two-operon approach involved a lower metabolic burden,
auguring for stable biocatalytic activities.